Biomedical subjects

E Smits

Publications and source records attributed to E Smits.

At least 19 recordsLinked to original sources

PubMed2004

RNA-based gene transfer for adult stem cells and T cells.

Electroporation of mRNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method. In this study, we investigated the potential of mRNA-based gene transfer to induce short-term transgene expression in adult stem cells and activated T cells, based on electroporation with mRNA encoding the enhanced green fluorescent protein. The results show efficient transgene expression in CD34-positive hematopoietic progenitor cells (35%), in in vitro cultured mesenchymal cells (90%) and in PHA-stimulated T cells (50%). Next to presentation of gene transfer results, potential applications of mRNA-based gene transfer in stem cells and T cells are discussed.

Adult↗

PubMed2001

Xyloside transport by XylP, a member of the galactoside-pentoside-hexuronide family.

This paper describes the functional characterization of the xyloside transporter, XylP, of Lactobacillus pentosus with the aid of a spectroscopy-based assay system. In order to monitor the transport reaction, the natural xyloside isoprimeverose, a building block of hemicellulose, and the analogue methyl-isoprimeverose were chemically synthesized by a new and efficient procedure. The XylP protein was purified by metal affinity chromatography, following high level expression in Lactococcus lactis from the nisin-inducible promoter. The purified XylP protein was incorporated into liposomes, in which the glucose dehydrogenase from Acinetobacter calcoaceticus (sGDH) was entrapped. sGDH can oxidize aldose sugars in the presence of dichlorophenol-indophenol as electron acceptor. The coupled assay thus involves XylP-mediated isoprimeverose uptake followed by internal oxidation of the sugar by sGDH, which can be monitored from the reduction of 2,6-dichlorophenol-indophenol at 600 nm. The uptake of isoprimeverose was stimulated by the presence of the non-oxidizable methyl-isoprimeverose on the trans-side of the membrane, indicating that exchange transport is faster than unidirectional downhill uptake. Unlike other members of the galactoside-pentoside-hexuronide family, XylP does not transport monosaccharides (xylose) but requires a glycosidic linkage at the anomeric carbon position. Consistent with a proton motive force-driven mechanism, the uptake was stimulated by a membrane potential (inside negative relative to outside) and inhibited by a pH gradient (inside acidic relative to outside). The advantages of the here-described transport assay for studies of carbohydrate transport are discussed.

Bacterial Proteins↗

PubMed2000

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells.

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

Amino Acid Sequence↗

PubMed2000

Adhesion receptor CD11b/CD18 contributes to neutrophil diapedesis across the bovine blood-milk barrier.

epithelium. Neutrophil migration across mammary arterial endothelial cells was almost completely dependent on CD18, the beta-chain of the beta(2) integrins, and to a lesser extent on CD11b, one of the alpha-chains of the beta(2) integrins. Neutrophil migration across collagen was partially blocked by monoclonal antibodies to CD18. No inhibition was observed by monoclonal antibodies to CD11b. Conversely, neutrophil diapedesis across mammary epithelial cells was dependent to a greater extent on CD11b. These results provide evidence for different CD11b/CD18-dependent mechanisms for neutrophil diapedesis across the various cell layers of the blood-milk barrier.

Animals↗

PubMed2000

Integration tutorials and seminars: a creative learning approach for occupational therapy curricula.

This paper, the first of two companion papers, describes a creative learning approach. Integration Tutorials and Seminars were developed to address concerns of regional fieldwork-education coordinators and preceptors about the ability of third-year student occupational therapists to integrate and apply academic and theoretical knowledge during fieldwork. This ability can be gained through experiential learning in the academic setting and is essential for the effective transfer of academic learning into the students' fieldwork-education experiences (Dale, 1994; Fidler, 1996; McCaugherty, 1991; Neistadt, 1996). The Modified Learning Model of Svinicki and Dixon (1987) was used as a template for an academic course fostering experiential learning. An important secondary goal was to nurture student self-directedness in learning using the philosophy of the Staged Self-Directed Learning Model (Grow, 1991). Case studies were used as a vehicle for engaging and challenging the students. The philosophy, guidelines and process of the Integration Tutorials and Seminars are presented and have the potential to be adapted for occupational therapy curricula around the globe.

Curriculum↗

PubMed2000

Integration tutorials and seminars: examining the integration of academic and fieldwork learning by student occupational therapists.

This paper, the second of two companion papers, describes a collaborative research study conducted by academic and fieldwork educators of an undergraduate educational programme in occupational therapy. The study examined the perception of student occupational therapists' ability to integrate and apply academic and theoretical knowledge in fieldwork education sites following the introduction of Integration Tutorials and Seminars to the curriculum. The literature review supports the need to develop educational strategies to improve this integration process. The qualitative research design consisted of guided interviews with preceptors and students during two consecutive fieldwork education experiences. Inductive data analysis revealed three themes that contribute to understanding students' ability to integrate academic learning during their fieldwork education: the fieldwork learning environment; preceptor influence on student learning; and the student learning process over time. The discussion section raises implications for academic and fieldwork education approaches.

Adult↗

PubMed2000

Cognitive rehabilitation: a pilot survey of therapeutic modalities used by Canadian occupational therapists with survivors of traumatic brain injury.

This pilot study used survey methodology to gather initial information from Canadian occupational therapists on 1) the delivery of cognitive rehabilitation services for survivors of traumatic brain injury and 2) the therapeutic approaches and modalities used currently to manage residual cognitive impairments. A questionnaire was developed, field-tested and mailed to 27 sample sites across Canada, selected non-randomly. Twenty returned questionnaires (74%) were included in the data pool. The findings revealed that a wide range of cognitive impairments were seen very often, with impairments in memory being most common. Respondents used both a cognitive remediation and compensatory approach and a combination of therapeutic modalities to address impairments of orientation, attention, and memory. There was an interesting and overlapping range of specific modalities identified. Inpatient and outpatient services were offered to survivors, primarily within the first year post-injury. These occupational therapists used various resources to develop expertise in cognitive rehabilitation. Possible implications of the pilot study findings for Canadian occupational therapists are high-lighted and discussed.

Adult↗

PubMed1999

The human homologue of Caenorhabditis elegans CED-6 specifically promotes phagocytosis of apoptotic cells.

A key feature of the process of programmed cell death (apoptosis) is the efficiency with which the dying cells are recognized and engulfed by phagocytes [1]. Apoptotic cells are rapidly cleared either by neighbouring cells acting as semi-professional phagocytes or by experts of the macrophage line, so that an inflammatory response is avoided [2]. The Caenorhabditis elegans gene ced-6 is required for efficient engulfment of apoptotic cells [3] and is one of a group of genes that define two partially redundant parallel pathways for the engulfment process [4] [5]. These pathways may be conserved across evolution, as two other engulfment genes have human homologues. A CED-5 homologue is part of a human CrkII-DOCK180-Rac signaling pathway proposed to mediate cytoskeletal reorganization [6] [7] [8] and a CED-7 homologue is similar to the ABC transporters [9] [10]. Here, we report the cloning and characterization of human CED-6, a human homologue of C. elegans CED-6. The 34 kDa hCED-6 protein is expressed in most tissues, some human cancer cells, and in primary human macrophages. We developed an assay that quantitates the phagocytic activity of mammalian macrophages: the number of apoptotic cells that have been internalized is measured by the uptake of lacZ-positive apoptotic cells by adherent transgenic macrophages. The results of this assay demonstrate that overexpression of hCED-6 promotes phagocytosis only of apoptotic cells and suggest that hCED-6 is the mammalian orthologue of C. elegans CED-6 and is a part of a highly conserved pathway that specifically mediates the phagocytosis of apoptotic cells.

Animals↗

PubMed1999

Effect of antibiotic induced bacterial clearance in the udder on L-selectin shedding of blood neutrophils in cows with Escherichia coli mastitis.

Bacterial clearance, L-selectin adhesion receptor expression and neutrophil recruitment during experimentally induced Escherichia coli mastitis were investigated. Acute mastitis was induced by injection of 10(4) cfu E. coli into both left quarters of 12 clinically healthy lactating cows, 2-6 weeks after parturition. Half the cows were treated intravenously 10 h after infection, and subcutaneously 30 h after infection with enrofloxacin. In both groups, bacterial count, milk serum albumin, milk somatic cell count, circulating leukocyte count and L-selectin expression on neutrophils were determined. Both groups responded to challenge with udder inflammation and fever. Treatment with enrofloxacin affected the elimination rate of bacteria at hours +14, +18, +24, +48, and +72 after challenge, but not the bacteriological cure rate (five out of six for treated and three out of six for nontreated cows). The drop in L-selectin expression found following challenge did not differ between groups throughout the study. No effect of treatment was found on SCC. Based on these findings, it may be concluded that when treatment is administered 10 h after infection, the number of bacteria in milk is not correlated with L-selectin expression on circulating PMN during experimentally induced E. coli mastitis. The initial bacterial load probably dictates the extent of the decrease in L-selectin expression and milk somatic cells.

Animals↗

PubMed1999

Diapedesis across mammary epithelium reduces phagocytic and oxidative burst of bovine neutrophils.

The effect of diapedesis on the phagocytic and oxidative burst activity of polymorphonuclear neutrophil (PMN) was examined, using an in vitro cell culture model consisting of a monolayer of primary mammary epithelial cells. Isolated blood PMN from 10 cows were added to the basal side of the epithelial cell monolayer. Diapedesis was induced by the addition of complement factor C5a to the apical side of the monolayer. PMN phagocytosis of Staphylococcus aureus and oxidative burst were measured before diapedesis on PMN that were non-activated and activated by incubation with C5a and on PMN after diapedesis, using flow cytometry. The percentages of PMN fluorescing due to phagocytosis of S. aureus and oxidative burst were reduced by 21.2 and 14.4%, respectively, after diapedesis. Pre-incubation in the presence of C5a had no effect on percentage PMN fluorescing due to phagocytosis or oxidative burst. The capacity for individual migrated PMN to phagocytose S. aureus and to produce an oxidative burst, as measured by the intensity of fluorescence, decreased by 34.2 and 30.3%. Activation of PMN with C5a increased intensity due to the oxidative burst, but had no effect on intensity due to phagocytosis. These data show that PMN diapedesis across mammary epithelium results in decreased phagocytosis and oxidative burst of the PMN.

Animals↗

PubMed1998

In vitro expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils during experimentally induced Streptococcus uberis mastitis.

The expression of adhesion receptors and diapedesis by polymorphonuclear neutrophils (PMN) were studied before and during experimentally induced Streptococcus uberis mastitis. Both quarters of the left half of the udders of five midlactation cows were inoculated with a suspension containing approximately 500 CFU of S. uberis 0140J. Clinical signs of an inflammatory reaction and leukocyte influx were observed 24 h after challenge. The expression of CD11b/CD18 adhesion receptors, determined by flow cytometry, was upregulated 24 h after challenge. A confluent monolayer of bovine secretory mammary epithelial cells on collagen-coated inserts was used to study PMN diapedesis. Bovine C5a was used as the chemoattractant. An 80% decrease in PMN diapedesis was observed 24 h after challenge. The decrease in diapedesis continued for 3 weeks after challenge.

Animals↗

PubMed1997

Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood.

Polymorphonuclear neutrophils (PMN) are important in host defence against bacterial infection in the bovine mammary gland and techniques are needed to evaluate their functional activities. A rapid and sensitive two-color flow cytometric method for simultaneous measurement of phagocytosis rate and oxidative burst activity of bovine PMN in small blood samples is described. The method utilizes the oxidation of intracellular dihydrorhodamine 123 to green fluorescent rhodamine 123 by oxidative burst products that were generated by incubating the PMNs with red fluorescent propidium iodide labeled Staphylococcus aureus. Phagocytosis and oxidative burst both increased with time of incubation and with increasing bacteria concentration. A 20 min phagocytosis time and a ratio of 25 bacteria to one cell were considered optimal for assaying bovine blood PMN activity. To further evaluate the proposed two-color flow cytometric method, blood samples were treated with factors known to interfere with phagocytosis and oxidative burst metabolism of human neutrophils. Incubation of whole bovine blood with cytochalasin B at 10 micrograms ml-1 resulted in an approximate 70% decrease in the phagocytosis rate with a concommitant decrease in oxidative burst activity. Staurosporine (2 micrograms ml-1) inhibited bovine neutrophil oxidative burst formation for 95% while the phagocytosis was unaffected. PMNs in whole blood samples of ten cows differed significantly in their ability to phagocytose Staphylococcus aureus and to produce reactive oxygen products. However, only a weak correlation was detected between the burst activity:phagocytosis ratio of ten individual cows as indicated by the ROD/PI index.

Animals↗

PubMed1996

Corticospinal input onto motor neurons projecting to ankle muscles in individuals with cerebral palsy.

Cross-correlograms between voluntarily active soleus (SOL) and tibialis anterior (TA) motor units were generated from seven control subjects and six subjects with spastic cerebral palsy (CP). Short-duration central peaks were observed in three subjects with spastic diplegia only. All subjects demonstrated reciprocal inhibition in TA following electrical stimulation of group I afferents to SOL, and all subjects with CP demonstrated strong activation of both TA and SOL in response to transcranial magnetic stimulation. Responses in SOL were stronger than those observed from controls. These data support the existence of abnormal corticospinal projections to soleus motor neurons in individuals with spastic CP. In spastic diplegia, short-term discharge synchrony between SOL and TA motor units may reflect abnormal interneuronal modulation at the spinal level. Abnormal corticospinal projections and/or modulation of spinal interneurons may contribute to the disordered movement patterns and co-activation observed in this population.

Ankle↗

PubMed1996

Cell culture system for studying bovine neutrophil diapedesis.

Neutrophils are the major defense against bacterial infection in the bovine mammary gland. Neutrophils migrate from blood into the lumen of the gland in response to inflammatory stimuli. This study describes the development of a system of cell culture that can be used to study neutrophil diapedesis through secretory and ductal mammary epithelial barriers. The culture system consists of successive layers of collagen, fibroblasts, collagen, and a confluent monolayer of secretory or ductal epithelial cells layered on a porous membrane. Confluence was determined by electrical resistance and trypan blue diffusion. Neutrophil diapedesis occurred from the basal to the apical surface of the monolayers. Purified complement C5a, fetal bovine serum that had been activated by zymosan, and fetal bovine serum that had been activated by Escherichia coli induced neutrophil diapedesis. Neutrophil diapedesis was greater across ductal cell monolayers. Blood neutrophils from five cows differed in their ability to migrate through the multilayered culture system in response to C5a. Monoclonal antibodies to C5a blocked diapedesis induced by purified C5a but had no effect on diapedesis induced by fetal bovine serum that had been activated by zymosan or by fetal bovine serum that had been activated by E. coli endotoxin, indicating that factors other than C5a were chemotactic for neutrophils. Monomeric IgG2, immune complexes, and E. coli endotoxin did not induce neutrophil diapedesis.

Animals↗

PubMed1994

Efficacy of seven retrograde tracers, compared in multiple-labelling studies of feline motoneurones.

The labelling efficacies of 7 retrograde tracers were evaluated following cut nerve exposure or intramuscular injection into the serially compartmentalized neck muscle, biventer cervicis. Tested tracers included Fast Blue (FB), Fluorogold (FG), dextran conjugated to fluorescein (FD), dextran conjugated to rhodamine (Fluororuby (FR), 3000 and 10,000 MW), fluorescent latex microspheres, horseradish peroxidase coupled to colloidal gold, and 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI). In 2 animals, horseradish peroxidase was also employed and spinal cords were processed for peroxidase activity to evaluate its effect on the appearance of cells labelled with fluorescent tracers. Four tracers, FB, FG, FD and FR, could be observed in motoneurones under the conditions of our study. FB and FG labelled comparable numbers of motoneurones following cut nerve exposure, but dissimilar numbers following intramuscular injection. FG diffused extensively following injection and was found in motoneurones not only in the appropriate ipsilateral segment but also adjacent ipsilateral and contralateral segments. Intramuscular injections of FB usually labelled fewer cells than cut nerve exposure, but evidence for spurious labelling following intramuscular injection could also be found. FD or FR labelled motoneurones following cut nerve exposure but not following intramuscular injection. The conjugated dextrans labelled more variable numbers of cells than FB or FG, but the labelled cells had similar patterns of distribution. The remaining tracers were ineffective as retrograde markers in our study, and the possible reasons for these failures are discussed.

Amidines↗

PubMed1994

The interobserver reproducibility of bioelectrical impedance analysis measurements in infants and toddlers.

Although nomograms to convert readings of resistance (R) and reactance (Xc) into estimates of body composition have begun to emerge for children as well as adults, there has been reluctance to use bioelectrical impedance analysis (BIA) in very young children due to the irritability of children, measurement instability, positioning of electrodes, and, of course, reproducibility of measurements. The precision of completely independent measurements of BIA indices was evaluated by two observers by comparing a series of 40 measurements of R and Xc and 36 measurements for weight in four malnourished children on 10 different days during the course of their nutritional recuperation. For weight, the CV (coefficient of variation) was 3.5% for observer A and 3.8% for observer B. There were no differences in the means across observers with respect to weight, R, and Xc. The results obtained in this study illustrate that properly trained observers following the same standards can produce equivalent data for BIA and weight measurements even in sick children.

Body Composition↗

PubMed1994

Organization of single motor units in feline sartorius.

1. We depleted single motor units in feline sartorius muscles of glycogen by stimulating their motoneurons intracellularly. We mapped the intramuscular distribution of depleted fibers by inspecting histological cross-sections throughout the length of sartorius. 2. We selected ten depleted motor units for detailed study and quantitative analysis. Nine motor units were located in the anterior head of sartorius. One was located in a muscle whose distal half appeared to have been damaged some time before the acute experiment. A single motor unit was located in the medial head of sartorius. 3. Five motor units were composed of fast-twitch glycolytic (FG) muscle fibers, two of fast-twitch oxidative glycolytic (FOG) muscle fibers, and three of slow-twitch oxidative (SO) muscle fibers. Estimates of the numbers of depleted fibers in motor units of anterior sartorius indicated that FG motor units were larger (mean 566 fibers) than FOG and SO motor units (SO mean 190, FOG mean 156 fibers). The SO motor unit in the damaged muscle had 550 fibers. One motor unit depleted in the medial head of sartorius had 270 fibers with FG profiles. 4. Muscle fibers belonging to each anterior motor unit were never distributed throughout the whole cross-section of anterior sartorius at any proximodistal level. Furthermore, fibers were distributed nonuniformly along the proximodistal axis of the muscle. In most muscles at least a few depleted fibers were found at all proximodistal levels. However, in one normal muscle and the damaged muscle, depleted fibers were confined to the proximal end. 5. The fibers in the medial motor unit were confined to a strip that did not extend across the whole cross-section of the muscle head. Fibers within this strip were scattered quite evenly from origin to insertion. This medial FG motor unit occupied a smaller territory and contained fewer fibers than anterior motor units of the same histochemical type. 6. These results show that sartorius motor units are not distributed uniformly in the mediolateral plane; those in anterior sartorius were distributed asymmetrically in the proximodistal axis as well. This finding has important functional implications for the way in which we model force development and transmission in sartorius and other long muscles.

Animals↗

PubMed1993

Synaptic mechanisms of cortical representational plasticity: somatosensory and corticocortical EPSPs in reorganized raccoon SI cortex.

1. Reorganizations of representational maps have been described for a variety of sensory and motor regions of cerebral neocortex in several species. The purpose of this study was to investigate synaptic mechanisms of the reorganizations of primary somatosensory cortex that follow removal of a digit or the joining of two digits into a syndactyly. We examined neurons in the cortical representation of digit 4 (d4). Intracellular recording was used to compare somatosensory and corticocortical excitatory postsynaptic potentials (EPSPs) in normal raccoons, with EPSPs recorded in two experimental groups of animals surviving for a mean of 22 wk after removal of d4, or union of d4 with digit 3 (d3). 2. In normal animals with d4 intact, EPSPs were evoked from this on-focus digit in 100% of cortical neurons. EPSPs were evoked from d3 and digit 5 (off-focus digits) in only a minority of neurons in normal raccoons. The incidence of somatosensory EPSPs from off-focus digits increased dramatically after removal of d4 or its union with d3. Latencies of EPSPs evoked from off-focus digits decreased after d4 removal, so that they were as short as latencies from d4 in normal animals. In contrast, for the group of animals with d3-d4 syndactyly, latencies of EPSPs from off-focus digits were not shorter than responses from these digits in normal animals. 3. Corticocortical EPSPs were no more common in animals with d4 removed than in intact animals. Furthermore, corticocortical EPSPs after d4 removal did not differ in their latencies, amplitudes, half-widths, or integrated amplitudes. The only detected change was that corticocortical EPSPs had faster rising phases after removal of d4. In contrast, after d3-d4 syndactyly, corticocortical EPSPs were more common than in normal animals. 4. Digit removal and digital syndactyly had distinctive effects on somatosensory and corticocortical EPSPs. These results do not identify unique synaptic mechanisms for cortical representational plasticity, nor do they specify the involved CNS site(s). Several synaptic mechanisms consistent with the results are considered in the DISCUSSION, including synaptic proliferation to form new synaptic connections and enhanced effectiveness of existing corticocortical synapses.

Afferent Pathways↗